Dairy cow behaviour in relation to health, welfare and milking
Animal welfare includes both physiological and mental health and is affected by several external and internal factors. In dairy cows, human care and the ability of the cows to cope with daily challenges are the most significant factors. The research presented in this dissertation focused on cow behaviour and aspects impacting on behavioural changes during mastitis and milking. To deepen knowledge of the relationship between the behaviour and health of cows and associated detection methods, we experimentally manipulated the health status of dairy cows through mastitis induction. Moreover, we tested and validated a thermal infrared camera for recording the udder skin temperature, which could be helpful in the case of early mastitis detection. Furthermore, we examined the relationship between cow behaviour and milking in herringbone and automatic milking systems. We established that cow behaviour changed during mastitis. The most apparent changes were in lying, eating and stepping behaviour. It was shown that inflammation affected the cow health status, which changed the behavioural priorities. In contrast to our expectations, visual signs in the udder and changes in milk composition occurred only 2 hours post-challenge, while clinical and behavioural changes were first recorded 4 hours post-challenge. However, changes in lying and restlessness behaviours were promising indicators for detecting signs in cows exposed to mastitis. The transient increase in body temperature of cows with experimentally-induced clinical mastitis was successfully detected by udder skin temperature detection with the help of a thermal camera. The udder skin temperature rose simultaneously with the rectal temperature. However, local inflammatory changes in the udder, appearing earlier than the rectal temperature increase, were not detected with udder skin temperature measurement by using the thermal infrared camera. Regarding cow behaviour as an indicator of the success and quality of the milking process, we found that half of the deviations occurring during automatic milking originated from cow behaviour, such as cows kicking, lifting their legs and moving during milking, and had their origins in machine failures. To conclude, cow behaviour can be used as an indicator for detection of mastitis and (un)successful milking. However, effectively functioning human animal technology interactions should be studied more in the future in order to enhance husbandry practices that can improve animal welfare.

Bioisosteric replacements are used in drug design during lead generation and optimization processes with the aim to replace one functional group of a known molecule by another while retaining biological activity. The reason to use bioisosteric replacements are typically to optimize bioavailability or reducing toxicity. Phosphate groups represent a paradigm to study bioisosteric replacements. Protein-phosphate interaction plays a critical role during molecular recognition processes, and for example kinases represent one of the largest families of drug targets. However, some challenges exclude phosphate as a promising lead-like building block: i) charged phosphates do not cross molecular membranes; ii) some widely expressed proteins such as phosphatases easily hydrolyze phosphoric acid esters, which lead phosphate-containing ligands to lose their binding affinities before reaching their biological targets; iii) introduction of phosphate groups to parent scaffold is not easy.
In the first part of the thesis work, I designed and implemented a computational protocol to mine information about phosphate structural replacements deposited in the Protein Data Bank. I constructed 116, 314, 271, and 42 sets of superimposed proteins where each set contains a reference protein to either POP, AMP, ADP, or ATP as well as a certain number of non-nucleotide ligands. 929 of such ligands are under study. The chemotypes that came out as structural replacements are diverse, ranging from common phosphate isosteres such as carboxyl, amide and squaramide to more surprising moieties such as benzoxaborole and aromatic ring systems. I exemplified some novel examples and interpreted the mechanism behind them. Local structural replacements are circumstance dependent: one chemical group valid in certain set-up cannot necessarily guarantee the success of another. The data from the study is available at http://86.50.168.121/phosphates_LSR.php.
In the second part, I synthesized fifteen compounds retaining the adenosine moieties and bearing bioisosteric replacements of the phosphate at the ribose 5'-oxygen to test their stability toward human macro domain protein 1. These compounds are composed of either a squaryldiamide or an amide group as the bioisosteric replacement and/or as a linker. To these groups a variety of substituents were attached: phenyl, benzyl, pyridyl, carboxyl, hydroxy and tetrazolyl. Biological evaluation using differential scanning fluorimetry showed that four compounds stabilized human MDO1 at levels comparable to ADP and one at level comparable to AMP. Virtual screening was also run to identify MDO1 binding ligands. Among 20,000 FIMM database lead-like molecules, 39 compounds were selected for testing and eleven compounds found active based on ADPr and Poly-ADPr competition binding assay. The assay is however not well validated and a second confirmatory assay was conducted using calorimetry. To the best of my knowledge, this is the first report of non-endogenous ligands of the human MDO1.
Altogether, this thesis highlights the versatility of molecular recognition processes that accompanies chemical replacements in compounds; this in turns shows the limits of the concepts of molecular similarity and classical bioisosterism that are based on the conservation of molecular interactions.

Salmonellosis is one of the most significant zoonoses worldwide and also in Finland. Salmonella Typhimurium definitive phage type (DT) 1 and Salmonella Infantis are considered endemic in Finland. These serovars have frequently caused outbreaks among humans. Salmonella Agona was not frequently encountered in Finland until a small outbreak among cattle farms occurred in 1994-1995.
Bacterial typing methods are used for outbreak investigations and for surveillance, where the data can be used for risk assessment calculations in addition to the future prevention of outbreaks.In particular the identification of factors that contribute to the persistence and spread of infection in endemic situations, estimations of the effect of animal reservoirs on human cases, and the identification of other risk factors for human infections are among the important reasons for typing.
Salmonella Infantis became more common in cattle in the 1980s, after it established itself in the broiler chicken production in Finland in 1971. In the 1990s, S. Infantis became the predominant serovar among cattle in Finland. In 1995, a feedborne outbreak of S. Infantis in cattle occurred. By XbaI-PFGE methodology we were able to identify the feedstuff-related genotype, which contained a 60 kb plasmid. Farms infected with the feedstuff-related genotype were also identified. The stability of the feedstuff-related genotype was followed on selected farms. The plasmid was stable on the farms during the follow-up period. The feedstuff-related genotype did not persist in the cattle population. Moreover, there was a general decline in bovine salmonella infections from 1997 onwards.
The genetic diversity of the S. Infantis isolates taken from Finnish cattle was also assessed. The S. Infantis infection in cattle is highly clonal as 99 per cent of the isolates had XbaI-PFGE profiles clonally related to each other. The major genotype was predominant both at the starting year of our analysis in 1985 and as the infection seemed to fade out in 2003. There is a trend towards less genetic diversity of the S. Infantis infection among domestic isolates from humans and poultry. The ribo/IS200-types and the most common XbaI-PFGE profiles determined among the analysed cattle isolates could also be detected among domestic isolates from poultry and humans. In our analysis of successive isolates from the same cattle herds, we frequently detected minor changes in banding patterns during long-lasting infections in individual herds. The sampling and testing of several isolates from a herd in outbreak investigations is therefore advisable.
In 1997, another small outbreak of Salmonella Agona among cattle farms occurred. The PFGE genotype of that outbreak was possibly related to that of the first outbreak, in 1994-1995. In 1999, a large outbreak of S. Agona of domestic origin involving more than 50 human cases occurred. Despite epidemiological investigations carried out by the local authorities, the source of the outbreak remained unknown. Based on our typing data, this outbreak was unrelated to the cattle farm outbreaks, though it did occur in the same region of Finland. The outbreak profile for the 1999 outbreak could not be found in any of the older isolates.
Salmonella Typhimurium DT1 has become the most common S. Typhimurium phage type among cattle farms in Finland, and has been detected annually since 1980. The infection is highly homogenous. The most common XbaI-PFGE profile can be seen in isolates from humans, domestic animals, hedgehogs and wild birds, so molecular subtyping by XbaI-PFGE alone is not discriminatory enough in analysing our endemic infection. A combination of XbaI-, BlnI-, and SpeI-PFGE can be applied in the analysis of outbreaks. Two clusters were formed by the combination of the XbaI-, BlnI-, and SpeI-PFGE profiles, IS200-profiles and possession of the serovar-specific virulence plasmid. The major cluster, typical of our endemic infection, had no virulence plasmid.

Because muscle glycogen has a crucial role as a substrate for energy metabolism, and because subsequent exercise performance may be dependent upon the extent of muscle glycogen resynthesis during recovery, post-exercise muscle glycogen repletion is very important for both human and equine athletes. Muscle glycogen repletion has been extensively studied in man, but due to the physiological differences between the two species, the results of human studies cannot be directly applied to horses.
The major finding of this study was the slow rate of resynthesis of glycogen, indicating that horses may be progressively depleted in muscle glycogen stores during an intensive training period.
The results emphasize the need for an adequate rest:work ratio over the training period to maintain a positive anabolic hormonal balance and to provide sufficient time for repletion of muscle glycogen stores.
When horses consume a normal basal diet, neither extra carbohydrate nor extra fat will enhance the repletion of muscle glycogen stores, but may, especially in unadapted horses, produce undesirable effects.
Maintaining horses in a good state of hydration seems to have a moderate positive effect on repletion of muscle glycogen stores. Providing horses with an isotonic glucose-electrolyte rehydration solution soon after exercise helps to overcome dehydration significantly better than providing them plain water.
In practical situations, changes in body weight serve as a useful indicator of recovery.

Yersinia pseudotuberculosis infections derive from ingestion of contaminated food or water. Typical symptoms of yersiniosis are fever and abdominal pain resulting from mesenteric lymphadenitis, and immunological sequelae are possible. The pathogen has recently caused several epidemics in Finland through fresh produce. However, the slow growth rate and poor competition of Y. pseudotuberculosis make its detection and isolation demanding. Polymerase chain reaction with primers targeted to virulence genes inv, virF, and yadA is thus often used in detection, although the sequence variability of the virulence genes is unknown. To study genetic variability of the virulence genes, inv, virF, and yadA of 18 Y. pseudotuberculosis strains, and two Yersinia similis strains originating from 12 different countries were sequenced. The greatest sequence variability was detected in yadA, while the variability of inv and virF was limited. The observed variability in yadA may hinder detection using PCR and also impact functional properties of YadA. Furthermore, the commonly used primers targeted to inv can, in addition to Y. pseudotuberculosis, detect Y. similis.
Y. pseudotuberculosis tolerates well low temperature and other stressful conditions in the environment and in the food chain. However, information on the stress tolerance mechanisms used by this pathogen is limited. Here, the roles of two-component systems (TCSs), alternative sigma factor σE, and RNA helicase CsdA of Y. pseudotuberculosis IP32953 under stress conditions were studied. The relative expression levels of 54 genes encoding putative TCSs in Y. pseudotuberculosis IP32953 were determined at 3°C and at the optimum growth temperature of 28°C. The relative expression levels of most of the genes were higher at 3°C than at 28°C, and TCS CheA/CheY encoding genes cheA and cheY had the highest relative expression levels at 3°C. Mutational analysis demonstrated the demand for cheA for optimal growth at 3°C. In addition, both cheA and cheY were required for motility. Increased expression of several TCS encoding genes demonstrate that probably in Y. pseudotuberculosis many TCSs play a role in adaptation to low temperatures. In addition, motility seems to be associated with cold tolerance.
The role of alternative sigma factor σE under stress conditions was studied by determining relative expression levels of rpoE encoding σE and using mutational analysis. Expression of rpoE was induced under low and high temperatures, acid and alkaline conditions, and osmotic and ethanol stress. Mutation of rpoE impaired or abolished growth at pH 5.0, at 3°C, at 37°C, at 42°C, and at 3% ethanol, demonstrating that functional σE is essential under several stress conditions in Y. pseudotuberculosis IP32953. In addition, the rpoE mutant had a higher minimum and a lower maximum growth temperature than the wild-type strain. Thus, in this pathogen, σE has a significant role in stress tolerance, and it contributes to survival during food processing and storage.
The function of a cold-induced RNA helicase CsdA has been unknown in Y. pseudotuberculosis. Investigation of the role of CsdA at 3°C by mutagenesis revealed that CsdA is essential for growth at low temperatures. At the optimum growth temperature of 28°C, no growth defect was seen. Also the minimum growth temperature of one of the mutants was significantly higher than that of the wild-type strain. Thus, CsdA enables the growth of Y. pseudotuberculosis in the food chain by allowing continuous growth at low temperatures. The results demonstrate that the foodborne pathogen Y. pseudotuberculosis counters environmental stress by using TCSs and alternative sigma factor, and by synthesizing cold-induced proteins.